Novel tricyclic pyrrolo-quinolines as pharmacological correctors of the mutant CFTR chloride channel

F508del, the most frequent mutation in cystic fibrosis (CF), impairs the stability and folding of the CFTR chloride channel, thus resulting in intracellular retention and CFTR degradation. The F508del defect can be targeted with pharmacological correctors, such as VX-809 and VX-445, that stabilize CFTR and improve its trafficking to plasma membrane. Using a functional test to evaluate a panel of chemical compounds, we have identified tricyclic pyrrolo-quinolines as novel F508del correctors with high efficacy on primary airway epithelial cells from CF patients. The most effective compound, PP028, showed synergy when combined with VX-809 and VX-661 but not with VX-445. By testing the ability of correctors to stabilize CFTR fragments of different length, we found that VX-809 is effective on the amino-terminal portion of the protein that includes the first membrane-spanning domain (amino acids 1–387). Instead, PP028 and VX-445 only show a stabilizing effect when the second membrane-spanning domain is included (amino acids 1–1181). Our results indicate that tricyclic pyrrolo-quinolines are a novel class of CFTR correctors that, similarly to VX-445, interact with CFTR at a site different from that of VX-809. Tricyclic pirrolo-quinolines may represent novel CFTR correctors suitable for combinatorial pharmacological treatments to treat the basic defect in CF.

All compounds were tested as correctors on CFBE41o-cells expressing F508del-CFTR and the halide-sensitive yellow fluorescent protein (HS-YFP). Cells were incubated for 24 h with compounds at 10 µM (Fig. 1A). As positive control, cells were separately incubated with the corrector VX-809 (1 µM). After treatment, compounds were removed and cells were acutely stimulated for 20-30 min with 20 µM forskolin (to increase cytosolic cAMP) plus 50 µM genistein (as a potentiator to further enhance CFTR channel opening). F508del-CFTR activity in the plasma membrane was evaluated by measuring the rate of HS-YFP quenching elicited by extracellular addition of an iodide-rich saline solution. Rescue of F508del-CFTR results in enhanced iodide influx and hence accelerated fluorescence quenching. Only one compound, later labeled as PP007, elicited a relatively modest increase in F508del-CFTR function corresponding to ~ 30% of VX-809 effect (Figs. 1A and 2). PP007 was retested at 1 and 10 µM in the presence and absence of 1 µM VX-809 (Fig. 1B). Activity was found at 10 but not at 1 µM. Importantly, the combination of PP007 (1 and 10 µM) with VX-809 improved F508del-CFTR function with respect to VX-809 alone. In particular, a near doubling of VX-809 rescue was observed when 10 µM PP007 was included. This interesting result prompted the synthesis and evaluation of a small set of PP007 analogs. A few compounds, having modifications at different positions of the scaffold, were synthesized and tested on CFBE41o-cells with the HS-YFP assay (Figs. 1C and 2). In particular, a bromine atom was introduced at position 3 (PP008), the p-methylbenzyl moiety at the pyrrole nitrogen was replaced by a methyl (PP010) or an unsubstituted benzyl (PP011) group, and the carbonyl of the pyridone moiety replaced by a methoxy group (PP014). Compounds PP008 and PP011 showed significant activity. Importantly, PP008 emerged as a very effective corrector able to markedly synergize with VX-809. We tested PP008 at multiple concentrations in the range 0.01-20 µM (Fig. 3A, top). A maximal rescue, corresponding to nearly 70% of VX-809 effect, was achieved at 10 µM with an EC 50 of ~ 1.78 µM. Interestingly, when tested in the presence of VX-809, the EC 50 was decreased to 1.05 µM (Fig. 3A, bottom). At 20 µM, PP008 caused a decrease in function with respect to 10 µM, either with and without VX-809 (Fig. 3A). www.nature.com/scientificreports/ We wondered whether PP008 has an effect on CFTR channel gating as a potentiator. Therefore, we incubated cells for 24 h with vehicle, with VX-809, or with VX-809 plus PP008. Then cells were acutely stimulated with forskolin, forskolin plus genistein, or forskolin plus PP008. In contrast to genistein, PP008 was unable to potentiate F508del-CFTR activity (Fig. 3B), irrespective of the type of corrector treatment.
The interesting properties of PP008 as a corrector encouraged us to test it in native airway epithelial cells. Figure 3C shows representative short-circuit current recordings on cultured bronchial epithelia from a F508del/ F508del patient. During these recordings, F508del-CFTR function was maximally stimulated with CPT-cAMP followed by the VX-770 potentiator and then inhibited with the selective CFTR inhibitor-172 (inh-172) 31  www.nature.com/scientificreports/ estimated from the amplitude of inhibitor effect. Figure 3D shows the results obtained from multiple preparations of bronchial and nasal epithelial cells of F508del/F508del patients. Cells treated with the combination of PP008 (10 µM) plus VX-809 showed significantly enhanced F508del-CFTR function with respect to cells treated with VX-809 alone. As shown in Fig. 3C, UTP was added at the end of experiments to elicit Ca 2+ -dependent Clsecretion, a process that involves activation of the TMEM16A Clchannel 32 . The size of the response to UTP was not affected by treatment with correctors, either PP008 or VX-809. Given the very positive results obtained with PP008, a campaign of chemical synthesis and functional evaluation was undertaken to explore the structure-activity relationships (SAR) within the chemical class in order to find correctors with improved potency and efficacy. A series of modifications were introduced into the scaffold of PP008. Replacement of the p-methylbenzyl moiety of PP008 with a m-methylbenzyl group (PP028; Fig. 2) resulted in a substantial improvement in potency and efficacy. The enlargement of the central ring to seven carbon atoms (PP048, PP055, and PP056; Fig. 2) led to loss of activity. A number of compounds were prepared bearing substituted benzyl moieties at the pyrrole nitrogen; in many cases, these analogues maintained good activity compared with PP028. The SAR studies showed that crucial structural features for maintaining a good corrector activity are the presence of the pyridin-2-one moiety bearing a benzenesulfonyl substituent and of a carboxyl ester group. Hydrolysis of the ester to the corresponding carboxylic acid or transformation into a carboxamide produced compounds devoid of activity.
We focused our subsequent validation and mechanistic studies on PP028 as one of the most effective correctors. In CFBE41o-cells, this compound increased anion transport in a dose-dependent way with an EC 50 of 1.1 µM (Fig. 4A, top). At the maximally-effective PP028 concentration, F508del-CFTR function was equivalent to that of cells treated with VX-809 alone. In the presence of VX-809, the effect of PP028 was largely amplified (Fig. 4A, bottom). In particular, the synergy generated by the combination of the two correctors elicited a nearly three-fold increase in anion transport with respect to each compound alone. Interestingly, as already seen with PP008, the EC 50 of PP028 was significantly decreased (from 1.1 to 0.5 µM) in the presence of VX-809.
To further validate the corrector activity of PP028, we evaluated its effect on F508del-CFTR protein. F508del-CFTR protein maturation and trafficking was investigated by determining its electrophoretic mobility (Fig. 4B). Under control conditions, F508del-CFTR mainly migrates as a single band of nearly 150 kDa, named band B, which corresponds to the immature partially-glycosylated form of the protein 19,20 . Treatment of cells with PP028, particularly in combination with VX-809, resulted in increased expression of a 180 kDa band, named as band C, which corresponds to the mature fully-glycosylated CFTR protein (Fig. 4B). PP008 was also effective but only in combination with VX-809 (Fig. 4B).
PP028 was tested in bronchial epithelial cells from F508del/F508del patients (Fig. 4C). The results confirmed our observations in the CFBE41o-cell line. The corrector was very effective in rescuing mutant CFTR function either alone and in combination with VX-809. Importantly, the effect of the two-corrector combination was an eight-fold increase in CFTR-dependent current with respect to vehicle-treated cells (Fig. 4C). As already observed for PP008, treatment of cells with PP028 did not alter Ca 2+ -dependent Clsecretion elicited by UTP.
We also investigated F508del-CFTR protein localization by immunofluorescence. We quantified CFTR signal in the perinuclear region and at the cell periphery. In vehicle-treated cells, the signal was weak and only localized in intracellular compartments (Fig. 5A). Treatment with VX-809 caused a significant increase in CFTR expression only in the perinuclear region (Fig. 5A,B). Instead, with PP028 treatment, perinuclear CFTR was not significantly increased but we noticed the appearance of a CFTR signal at the cell periphery, consistent with plasma membrane localization (Fig. 5A). Accordingly, the ratio of peripheral to perinuclear signal was significantly increased by PP028 (Fig. 5C). The peripheral signal was more evident by treating cells with the PP028/ VX-809 combination (Fig. 5A).
We also evaluated VX-445 14 , a second generation Vertex corrector that has been included in the triple combination of CFTR modulators, recently approved under the brand name Trikafta/Kaftrio 33 . Combination of VX-445 and VX-809 generated a high extent of synergy, comparable to that obtained with VX-809/4172 and VX-809/ PP028 (Fig. 6C). No additivity/synergy was instead observed when VX-445 was combined with 4172 or PP028.
We tested the ability of PP028 to act synergistically with VX-661, which is the C1 corrector replacing VX-809 in the Symdeko/Symkevi and Trikafta/Kaftrio drug combinations. For these experiments, we also included chronic treatment with/without VX-770. Addition of PP028 significantly amplified the rescue by VX-661 (Fig. 6D). Inclusion of VX-770 did not change the effect of PP028/VX-661 combination. The results obtained with PP028/VX-661 were comparable with those obtained with VX-445/VX-661 (Fig. 6D). www.nature.com/scientificreports/ We evaluated the possibility of cytotoxic effects elicited by PP028 treatment. CFBE41o-cells, plated at subconfluent density, were treated with PP028 at multiple concentrations in the range 0.625-20 µM. VX-445 and doxorubicin were also tested for comparison. After 24 h, cell number was quantified by counting the nuclei stained with NucBlue. We found no dose-dependent decrease in cell number with either PP028 or VX-445 ( Fig. 6E) thus indicating lack of clear cytotoxic effects. In contrast, doxorubicin, as a control cytotoxic compound, markedly decreased cell number in a dose-dependent way (Fig. 6E).
To investigate the site of action of PP028, we tested its ability to stabilize different fragments of CFTR protein, an assay that has been previously adopted to study other correctors 22,34,35 . PP028 activity was compared with that of VX-809 and VX-445. We first tested these three correctors on MSD1 fragment (amino acids: 1-387). By immunoblot, we detected two bands of 37 and 30 kDa, respectively (Fig. 7A). The upper band corresponds to the expected molecular size of the MSD1 fragment. The lower band may result from a proteolytic cleavage. We considered the 37 kDa band for densitometric analysis. As shown in Fig. 6A, VX-809, but not PP028 or VX-445, stabilized the MSD1 fragment, as evident from the increased band intensity. PP028 and VX-445 were not effective even if combined with VX-809 (Fig. 7A). We then tested the three correctors on MSD1-NBD1 fragment containing the F508del mutation (amino acids: 1-633). Expression of this construct resulted in a single band www.nature.com/scientificreports/ of the expected molecular size. MSD1-NBD1(∆F) was sensitive to VX-809 (Fig. 7B), but showed no significant stabilization by PP028 and VX-445, either alone or in combinations. Comparable results were also obtained with the fragment including the R domain, i.e. MSD1-NBD1(∆F)-R (amino acids: 1-823) (Fig. 7C). Finally, we tested correctors on the MSD1-NBD1(F508del)-R-MSD2 (amino acids: 1-1181; Fig. 7D). This construct, which includes most of CFTR domains except for NBD2 and the carboxy-terminus, was responsive to all three correctors. More precisely, PP028 was not effective by itself but significantly increased band intensity in combination with VX-809 compared to VX-809 alone (Fig. 7D). VX-445 was effective as a single treatment compared to vehicle and markedly enhanced fragment stability when combined to VX-809.

Discussion
The identification of small molecules able to improve the maturation, trafficking, and gating of mutant CFTR is of high relevance to design pharmacological strategies to treat the basic defect in CF. In the present study, we have identified a novel class of correctors. The structure of angular furocumarins and constrained bithiazoles, encouraged the evaluation of several heteroanalogues of an in house library, which was the starting point of our chemical exploration. By screening our focused library, we found PP007 as the only active compound, with the interesting property of showing synergy when combined with VX-809, a typical C1 corrector 22 . Starting from PP007, we generated a series of analogues that led us first to PP008, a close analogue of hit PP007, having improved potency and efficacy on F508del-CFTR as corrector in cell lines and primary airway epithelial cells. Further modifications of the structure of PP008 allowed us to discover PP028, a derivative with a better activity profile than the parent compound. In the presence of VX-809, PP008 and PP028 do not only show enhanced efficacy but also an improvement in potency as indicated by the decrease in the EC 50 value. These results suggest that VX-809 and PP compounds cooperate by acting on two different sites of CFTR protein. We combined PP028 with other types of correctors, including 3151, as a C2 corrector, and 4172 and VX-445, as C3 correctors 22,23 . We found synergy with 3151 but not with 4172 and VX-445. Actually, we found a sort of antagonism between PP028 and 4172, suggesting that the binding of one molecule negatively affects the binding and/or efficacy of the other molecule. The lack of additivity/synergy between PP028, VX-445, and 4172 indicates that all of them belong to the same class of correctors. Previously, VX-445 and 4172 were classified as C3 correctors, with a possible mechanism of action involving binding to NBD1 22,23 . Therefore, according to our functional data, PP028 should also be a C3 corrector. Recently, VX-809 binding site was identified in MSD1 21 , in agreement with previous studies showing that VX-809 improves the stability of a CFTR fragment that only includes the first six transmembrane helices 34 . www.nature.com/scientificreports/ We have adopted the stabilization of CFTR fragments of different length as an assay to investigate the mechanism of action of PP028 in comparison with other CFTR correctors. As expected, VX-809 was the only compound that markedly stabilized MSD1. Surprisingly, VX-445 did not stabilize the MSD1-NBD1(∆F) fragment, despite previous indications, obtained with other types of assays, that VX-445 binds to NBD1. PP028 was also ineffective on the same fragment, as well as on MSD1-NBD1(∆F)-R. Instead, we found a stabilizing effect of PP028 and VX-445 in combination with VX-809, when MSD2 was included in the CFTR construct. VX-445 was also effective in the absence of VX-809. These results could imply that the binding site of C3 correctors is in the second transmembrane domain of CFTR. While finalizing our manuscript, a cryo-EM study was published showing that VX-445 indeed interacts with MSD2, more precisely with amino acid residues (Trp1098, Arg1102) of transmembrane helix 11 36 . Intriguingly, VX-445 does not bind to a well defined pocket inside CFTR but interacts with the surface of transmembrane region. Furthermore, a small portion of VX-445 also interacts with the lasso domain (Ser18, Arg21), which belongs to the CFTR amino-terminus 36 . Therefore, it appears that VX-445 forms a bridge between two distinct CFTR regions, a type of interaction that may be important for protein stabilization. It will be interesting to assess if PP compounds share the same binding site of VX-445.
In conclusion, our study has revealed a new family of CFTR correctors, showing a marked efficacy in primary airway epithelial cells from CF patients with F508del mutation. Considering that PP028, the best corrector of this family, was found within a relatively small set of analogues, we can postulate that further exploration of the chemical space around the PP scaffold, also supported by the structural information about the binding site of C3 correctors 36 , may lead to compounds with improved ability to rescue F508del and other CFTR mutants with trafficking defects. This is an important step since the potency and efficacy of PP compounds on mutant CFTR function and trafficking needs to be improved.

Materials and methods
Chemistry. Compounds shown in Fig. 2 were prepared according to our previously reported methods 37 .
Human bronchial epithelial cells were obtained from the "Servizio Colture Primarie", a service of Italian Foundation for Cystic Fibrosis. The collection of human bronchi for scientific purposes was approved by the relevant Ethical Committee (Comitato Etico Regione Liguria; registration number: ANTECER 042-09/07/2018 and CER 28/2020). The protocol for collection and culture of human bronchial epithelial cells was described in detail in a previous study 37 . Briefly, human bronchi, dissected from the lungs of CF patients undergoing lung transplant, were washed and incubated overnight at 4 °C in protease XIV solution. Epithelial cells were then detached by vigorously pipetting of bronchial lumen, pelleted by centrifugation, and dissociated by 5-10 min treatment with trypsin 38 . After neutralization of trypsin with complete DMEM/F12 medium, cells were centrifuged and resuspended in a serum-free medium. This medium contained a 1:1 mixture of LHC basal medium and RPMI 1640 (Thermo Fisher Scientific) plus hormones, growth factors, and other supplements as indicated previously 37 . Additionally, to promote the proliferation of basal stem cells 39 , the medium was supplemented with bone morphogenetic protein (BMP) antagonist (DMH-1, 1 µM; Tocris), transforming growth factor-β (TGF-β) antagonist (A 83-01, 1 µM; Tocris), and the rho-associated protein kinase 1 (ROCK1) inhibitor (Y-27632, 10 µM; Tocris). After 4-5 passages, cells were seeded (on Snapwell porous inserts (cc3801, Corning Costar; 500,000 cells per insert). After 24 h from seeding, the medium was switched to a 1:1 mixture of DMEM and Ham's F12 plus 2% New Zealand Fetal Bovine Serum (Thermo Fisher Scientific) plus hormones, and supplements as previously indicated 37 . The medium was replaced daily on both sides of permeable supports for 7 days. Subsequently, the apical medium was totally removed and the medium was only maintained, with daily change, on the basolateral side (air-liquid interface condition). Cells were maintained under this condition for 2 weeks before carrying out the experiments.

HS-YFP assay.
CFBE41o-cells co-expressing F508del-CFTR and HS-YFP were plated at high density (35,000 cells/well) in black-wall clear-bottom 96-well microplates (cc3603 Corning). After 24 h, cells were treated for further 24 h with vehicle or compounds at various concentrations in complete culture medium. After final treatment, each well in the microplate was washed three times with 200 µl complete PBS. After washing, each well received 60 µl of an activating solution containing 20 µM forskolin and 50 µM genistein in PBS. In some experiments, genistein was omitted or replaced by PP008 (Fig. 3B) or replaced by 1 µM VX-770 (Fig. 6D). Cells were stimulated with this solution for 30 min. Then the microplate was transferred to a FLUOstar Omega microplate reader (BMG LABTECH) equipped with syringe pumps and excitation/emission filters optimized for Enhanced Yellow Fluorescent Protein, EYFP (ET500/20 × and ET535/30 m, respectively; Chroma Technology Corporation). The assay was done in "well mode", which consisted in a continuous fluorescence reading of 14 s for each well (0.2 s sampling time, 10 flashes/sample). At 2 s from start, the syringe pump injected 165 µl of a modified PBS in which NaCl was replaced with NaI (170 µl/s flow rate). The fluorescence recording from each well was background substracted and then normalized for the initial value. The fluorescence decay resulting from Iinflux and HS-YFP quenching was fitted with an exponential function to derive the maximum quenching rate (dF/dt). This calculation was done using a procedure compiled in the Igor Pro software (WaveMetrics, Lake Oswego, OR, USA). The mean CFTR fluorescence intensity signal was measured with Fiji (NIH) software in regions of interests (ROIs) placed in two different sites of the cell, perinuclear and peripheral. The perinuclear region was clearly visible because of nuclear DAPI staining. For the peripheral region, a localization compatible with CFTR trafficking to plasma membrane, we placed the ROI at the cell edge, identified in phase contrast images. Ten cells per condition were analyzed. For each cell, six measurements of perinuclear and peripheral fluorescence intensity were done and the results were averaged. Data are presented as perinuclear signal and ratio of peripheral/ perinuclear signals.

Scientific Reports
Immunoblot analysis of full length CFTR. CFBE41o-cells co-expressing F508del-CFTR and HS-YFP, plated on 6-well microplates, were treated for 24 h with correctors or vehicle and then lysed with a buffer containing 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM PMSF and complete protease inhibitor cocktail (Roche). Cell lysates were subjected to centrifugation at 14,500 rpm at 4 °C for 15 min. Protein concentration was determined with the BCA assay (Euroclone) following the manufacturer's instructions and using bovine serum albumin as the standard. Equal amounts of proteins (20 μg) were separated on 4-15% mini protean TGX Precast gels (Bio-Rad laboratories, Hercules, CA, USA), transferred to a nitrocellulose membrane with Trans-Blot Turbo system (Bio-Rad Laboratories Inc., Hercules, CA, USA). The membrane was incubated overnight at 4 °C with an anti-CFTR rabbit primary antibody (D6W6L, Cell Signaling Technology, USA) diluted 1:1000 in Tris-buffered saline with 0.5% Tween-20 (TBST) plus 5% skimmed-milk. The membrane was then washed three times and incubated for 1 h with a polyclonal goat anti-rabbit antibody conjugated with horseradish peroxidase (Dako) diluted 1:10,000 in TBST plus 5% milk. Immunodetection was subsequently visualized by chemiluminescence using the SuperSignal West Femto Substrate (Thermo Fisher Scientific). For quantification, images were analyzed with the Image Quant TL software (GE Healthcare). For each lane, intensity of signal was measured in two regions of interest, corresponding to bands B and C (150 and 180 kDa). Data were normalized using the GAPDH loading control. www.nature.com/scientificreports/ protein (20 μg) were separated on 4-15% mini protean TGX Precast gels (Bio-Rad laboratories, Hercules, CA, USA) and transferred to nitrocellulose membranes with Trans-Blot Turbo system (Bio-Rad Laboratories Inc., Hercules, CA, USA). Membranes were blocked with 5% skimmed-milk in TBST for two hours at room temperature and then incubated over night at 4 °C in agitation with the primary antibody. For fragments MSD1, MSD1-NBD1(∆F), and MSD1-NBD1(∆F)-R we used mouse anti-CFTR monoclonal antibody, clone MM13-4 (Sigma-Aldrich). For the MSD1-NBD1(∆F)-R-MSD2 fragment, we used the rabbit anti-CFTR D6W6L antibody (Cell Signaling Technology). Primary antibodies were diluted 1:1000 in TBST plus 5% skimmed-milk. Membranes were then incubated for one hour at room temperature with anti-mouse or anti-rabbit secondary antibodies conjugated to horseradish peroxidase (Dako, Agilent) diluted 1:2000) in TBST plus 5% skimmed-milk. After CFTR fragment detection, membranes were incubated with primary antibodies for GAPDH (for MSD1, MSD1-NBD1(∆F)-R, and MSD1-NBD1(∆F)-R-MSD2) or beta-actin (for MSD1-NBD1(∆F)). For GAPDH, we used the mouse MAB374 clone 65C antibody (Sigma-Aldrich). For beta-actin, we used the mouse A1978 antibody (Sigma-Aldrich). Both antibodies were diluted 1:10,000 and 1:2000, respectively, in TBST plus 5% skimmed-milk. For GAPDH detection, membranes were previously stripped with the Restore Western Blot Stripping Buffer (Thermo Fisher Scientific). The HRP-conjugated anti-mouse secondary antibody was used at dilution 1:20,000 for beta-actin and 1:50,000 for GAPDH. Proteins were visualized by chemiluminescence with the SuperSignal West Femto Substrate (Thermo Fisher Scientific). Images were obtained using the Molecular Imager UVITEC Cambridge System and subsequently analysed with Fiji software (National Institutes of Health, Bethesda, MD, USA). CFTR band intensities were analyzed as regions of interest and normalized against the GAPDH or beta-actin control.

Immunoblot analysis of CFTR fragments.
Cytotoxicity assay. CFBE41o-cells were seeded in 96-well plates (PhenoPlate, PerkinElmer) at a density of 20,000 cells per well. After 24 h, cells were treated for further 24 h with PP028, VX-445, and doxorubicin at various concentrations. Finally, cells were labeled with NucBlue (Invitrogen, Live Cell Stain ReadyProbes reagent). The images were acquired with the High Content Analysis System, OperettaCLS (PerkinElmer) and analyzed by Harmony Software (PerkinElmer).

Statistical analysis and data visualization.
Data are shown as scatter dot plots plus mean ± SD or as representative images. Each symbol in the scatter dot plots represents the result of an independent experiment. To assess statistically significant difference between groups of data, we first used the Kolmogorov-Smirnov test to assess normal distribution. For normally distributed data, we then used ANOVA followed by Tukey's post hoc test. For data with non-normal distribution, we used Kruskal-Wallis with Dunn's non parametric test. Statistical analysis was done with PRISM software (GraphPad). All graphs and figures were prepared with Igor Pro (WaveMetrics).
Ethics approval and consent to participate. The collection and study of bronchial and nasal epithelial cells from human subjects was done according to the guidelines of the Declaration of Helsinki and approved by the "Comitato Etico Regione Liguria" (Registration Numbers: ANTECER, 042-09/07/2018 and CER 28/2020). Informed consent to participate in scientific studies and to publish data in research journal articles was also obtained from all subjects involved in the study. www.nature.com/scientificreports/ Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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